Aaron J. Mercer

Inhibition of Glycogen Synthase Kinase 3β (GSK3β) Decreases Inflammatory Responses in Brain Endothelial Cells

Immune mediators and leukocyte engagement of brain microvascular endothelial cells (BMVECs) contribute to blood-brain barrier impairment during neuroinflammation. Glycogen synthase kinase 3beta (GSK3beta) was recently identified as a potent regulator of immune responses in in vitro systems and animal models. However, the role of GSK3beta in regulation of immune endothelial functions remains undetermined. Here we evaluated the effect of GSK3beta inhibition on the regulation of inflammatory responses in BMVECs. A focused PCR gene array of 84 genes was performed to identify the cytokine and chemokine gene expression profile in tumor necrosis factor (TNF) alpha-stimulated BMVECs after GSK3beta inactivation by specific inhibitors. Fifteen of 39 genes induced by TNFalpha stimulation were down-regulated after GSK3beta inhibition. Genes known to contribute to neuroinflammation that were most negatively affected by GSK3beta inactivation included IP-10/CXCL10, MCP-1/CCL2, IL-8/CXCL8, RANTES/CCL5, and Groalpha/CXCL1. GSK3beta suppression resulted in diminished secretion of these proinflammatory mediators by inflamed BMVECs detected by ELISA. GSK3beta inhibition in BMVECs reduced adhesion molecule expression as well as monocyte adhesion to and migration across cytokine stimulated BMVEC monolayers. Interactions of monocytes with TNFalpha-activated BMVECs led to barrier disruption, and GSK3beta suppression in the endothelium restored barrier integrity. GSK3beta inhibition in vivo substantially decreased leukocyte adhesion to brain endothelium under inflammatory conditions. In summary, inhibition of GSK3beta emerges as an important target for stabilization of the blood-brain barrier in neuroinflammation.

Lateral Mobility of Presynaptic L-Type Calcium Channels at Photoreceptor Ribbon Synapses

At most synapses, presynaptic Ca2+ channels are positioned near vesicle release sites, and increasing this distance reduces synaptic strength. We examined the lateral membrane mobility of presynaptic L-type Ca2+ channels at photoreceptor ribbon synapses of the tiger salamander (Ambystoma tigrinum) retina. Movements of individual Ca2+ channels were tracked by coupling quantum dots to an antibody against the extracellular α2δ4 Ca2+ channel subunit. α2δ4 antibodies labeled photoreceptor terminals and colocalized with antibodies to synaptic vesicle glycoprotein 2 and voltage-gated Ca2+ channel 1.4 (CaV1.4) α1 subunits. The results show that Ca2+ channels are dynamic and move within a confined region beneath the synaptic ribbon. The size of this confinement area is regulated by actin and membrane cholesterol. Fusion of nearby synaptic vesicles caused jumps in Ca2+ channel position, propelling them toward the outer edge of the confinement domain. Channels rebounded rapidly toward the center. Thus, although CaV channels are mobile, molecular scaffolds confine them beneath the ribbon to maintain neurotransmission even at high release rates.

The dynamic architecture of photoreceptor ribbon synapses: cytoskeletal, extracellular matrix, and intramembrane proteins.

Rod and cone photoreceptors possess ribbon synapses that assist in the transmission of graded light responses to second-order bipolar and horizontal cells of the vertebrate retina. Proper functioning of the synapse requires the juxtaposition of presynaptic release sites immediately adjacent to postsynaptic receptors. In this review, we focus on the synaptic, cytoskeletal, and extracellular matrix proteins that help to organize photoreceptor ribbon synapses in the outer plexiform layer. We examine the proteins that foster the clustering of release proteins, calcium channels, and synaptic vesicles in the presynaptic terminals of photoreceptors adjacent to their postsynaptic contacts. Although many proteins interact with one another in the presynaptic terminal and synaptic cleft, these protein-protein interactions do not create a static and immutable structure. Instead, photoreceptor ribbon synapses are remarkably dynamic, exhibiting structural changes on both rapid and slow time scales.

Location of Release Sites and Calcium-Activated Chloride Channels Relative to Calcium Channels at the Photoreceptor Ribbon Synapse

Vesicle release from photoreceptor ribbon synapses is regulated by L-type Ca(2+) channels, which are in turn regulated by Cl(-) moving through calcium-activated chloride [Cl(Ca)] channels. We assessed the proximity of Ca(2+) channels to release sites and Cl(Ca) channels in synaptic terminals of salamander photoreceptors by comparing fast (BAPTA) and slow (EGTA) intracellular Ca(2+) buffers. BAPTA did not fully block synaptic release, indicating some release sites are <100 nm from Ca(2+) channels. Comparing Cl(Ca) currents with predicted Ca(2+) diffusion profiles suggested that Cl(Ca) and Ca(2+) channels average a few hundred nanometers apart, but the inability of BAPTA to block Cl(Ca) currents completely suggested some channels are much closer together. Diffuse immunolabeling of terminals with an antibody to the putative Cl(Ca) channel TMEM16A supports the idea that Cl(Ca) channels are dispersed throughout the presynaptic terminal, in contrast with clustering of Ca(2+) channels near ribbons. Cl(Ca) currents evoked by intracellular calcium ion concentration ([Ca(2+)](i)) elevation through flash photolysis of DM-nitrophen exhibited EC(50) values of 556 and 377 nM with Hill slopes of 1.8 and 2.4 in rods and cones, respectively. These relationships were used to estimate average submembrane [Ca(2+)](i) in photoreceptor terminals. Consistent with control of exocytosis by [Ca(2+)] nanodomains near Ca(2+) channels, average submembrane [Ca(2+)](i) remained below the vesicle release threshold (∼ 400 nM) over much of the physiological voltage range for cones. Positioning Ca(2+) channels near release sites may improve fidelity in converting voltage changes to synaptic release. A diffuse distribution of Cl(Ca) channels may allow Ca(2+) influx at one site to influence relatively distant Ca(2+) channels.

Release from the cone ribbon synapse under bright light conditions can be controlled by the opening of only a few Ca(2+) channels.

Light hyperpolarizes cone photoreceptors, causing synaptic voltage-gated Ca(2+) channels to open infrequently. To understand neurotransmission under these conditions, we determined the number of L-type Ca(2+) channel openings necessary for vesicle fusion at the cone ribbon synapse. Ca(2+) currents (I(Ca)) were activated in voltage-clamped cones, and excitatory postsynaptic currents (EPSCs) were recorded from horizontal cells in the salamander retina slice preparation. Ca(2+) channel number and single-channel current amplitude were calculated by mean-variance analysis of I(Ca). Two different comparisons-one comparing average numbers of release events to average I(Ca) amplitude and the other involving deconvolution of both EPSCs and simultaneously recorded cone I(Ca)-suggested that fewer than three Ca(2+) channel openings accompanied fusion of each vesicle at the peak of release during the first few milliseconds of stimulation. Opening fewer Ca(2+) channels did not enhance fusion efficiency, suggesting that few unnecessary channel openings occurred during strong depolarization. We simulated release at the cone synapse, using empirically determined synaptic dimensions, vesicle pool size, Ca(2+) dependence of release, Ca(2+) channel number, and Ca(2+) channel properties. The model replicated observations when a barrier was added to slow Ca(2+) diffusion. Consistent with the presence of a diffusion barrier, dialyzing cones with diffusible Ca(2+) buffers did not affect release efficiency. The tight clustering of Ca(2+) channels, along with a high-Ca(2+) affinity release mechanism and diffusion barrier, promotes a linear coupling between Ca(2+) influx and vesicle fusion. This may improve detection of small light decrements when cones are hyperpolarized by bright light.

Peroxisome proliferator-activated receptor-γ activation suppresses Hiv-1 replication in an animal model of encephalitis

Objective:Poor penetration of antiretroviral therapy across the blood–brain barrier poses an impediment on control of HIV-1 infection in brain macrophages. Peroxisome proliferator-activated receptor (PPAR)-γ, a member of the nuclear receptors family, regulates important physiological functions (including anti-inflammatory effects) in response to ligand-mediated activation. As PPARγ agonists are rapidly absorbed by oral administration and efficiently permeate the blood–brain barrier, we hypothesized that PPARγ stimulation may suppress HIV-1 replication. Design and methods:We investigated the effect of PPARγ ligand (rosiglitazone) on HIV-1 replication in human monocyte-derived macrophages and in vivo using a murine model (immunodeficient mice reconstituted with human lymphocytes and intracerebrally inoculated with HIV-1 infected macrophages) of HIV-1 encephalitis. Results:Treatment with rosiglitazone caused a significant decrease of virus infection in macrophages. PPARγ stimulation inhibited virus replication by modulating NF-κB activation in a receptor-dependent manner, leading to downregulation of HIV-1 long terminal repeat (LTR) promoter activity and suppression of HIV-1 replication. These effects were PPARγ specific as PPARγ silencing or addition of PPARγ antagonist abolished effects of PPARγ stimulation on HIV-1 LTR and virus replication. Using a murine model for HIV-1 encephalitis, we demonstrated that PPARγ ligand suppressed HIV-1 replication in macrophages in brain tissue and reduced viremia by 50%. Conclusion:In vitro data delineated the novel mechanism by which PPARγ activation suppresses HIV-1 replication, and in vivo findings underscored the ability of PPARγ agonists to reduce HIV-1 replication in lymphocytes and brain macrophages, thus offering a new therapeutic intervention in brain and systemic infection.

Regulation of presynaptic strength by controlling Ca2+ channel mobility: effects of cholesterol depletion on release at the cone ribbon synapse

Synaptic communication requires proper coupling between voltage-gated Ca(2+) (Ca(V)) channels and synaptic vesicles. In photoreceptors, L-type Ca(V) channels are clustered close to synaptic ribbon release sites. Although clustered, Ca(V) channels move continuously within a confined domain slightly larger than the base of the ribbon. We hypothesized that expanding Ca(V) channel confinement domains should increase the number of channel openings needed to trigger vesicle release. Using single-particle tracking techniques, we measured the expansion of Ca(V) channel confinement domains caused by depletion of membrane cholesterol with cholesterol oxidase or methyl-β-cyclodextrin. With paired whole cell recordings from cones and horizontal cells, we then determined the number of Ca(V) channel openings contributing to cone Ca(V) currents (I(Ca)) and the number of vesicle fusion events contributing to horizontal cell excitatory postsynaptic currents (EPSCs) following cholesterol depletion. Expansion of Ca(V) channel confinement domains reduced the peak efficiency of release, decreasing the number of vesicle fusion events accompanying opening of each Ca(V) channel. Cholesterol depletion also inhibited exocytotic capacitance increases evoked by brief depolarizing steps. Changes in efficiency were not due to changes in I(Ca) amplitude or glutamate receptor properties. Replenishing cholesterol restored Ca(V) channel domain size and release efficiency to control levels. These results indicate that cholesterol is important for organizing the cone active zone. Furthermore, the finding that cholesterol depletion impairs coupling between channel opening and vesicle release by allowing Ca(V) channels to move further from release sites shows that changes in presynaptic Ca(V) channel mobility can be a mechanism for adjusting synaptic strength.

Intracellular pH modulates inner segment calcium homeostasis in vertebrate photoreceptors

Neuronal metabolic and electrical activity is associated with shifts in intracellular pH (pH(i)) proton activity and state-dependent changes in activation of signaling pathways in the plasma membrane, cytosol, and intracellular compartments. We investigated interactions between two intracellular messenger ions, protons and calcium (Ca²(+)), in salamander photoreceptor inner segments loaded with Ca²(+) and pH indicator dyes. Resting cytosolic pH in rods and cones in HEPES-based saline was acidified by ∼0.4 pH units with respect to pH of the superfusing saline (pH = 7.6), indicating that dissociated inner segments experience continuous acid loading. Cytosolic alkalinization with ammonium chloride (NH₄Cl) depolarized photoreceptors and stimulated Ca²(+) release from internal stores, yet paradoxically also evoked dose-dependent, reversible decreases in [Ca²(+)](i). Alkalinization-evoked [Ca²(+)](i) decreases were independent of voltage-operated and store-operated Ca²(+) entry, plasma membrane Ca²(+) extrusion, and Ca²(+) sequestration into internal stores. The [Ca²(+)](i)-suppressive effects of alkalinization were antagonized by the fast Ca²(+) buffer BAPTA, suggesting that pH(i) directly regulates Ca²(+) binding to internal anionic sites. In summary, this data suggest that endogenously produced protons continually modulate the membrane potential, release from Ca²(+) stores, and intracellular Ca²(+) buffering in rod and cone inner segments.